Restoring muscle function to patients with degenerative muscle diseases such as Duchenne Muscular Dystrophy (DMD) remains a significant clinical problem. In DMD, the increased susceptibility of muscle to damage, due to a dystrophin mutation, combined with exhaustion of the stores of resident cells capable of regenerating damaged areas cause extensive replacement of contractile material with connective tissue and fat, decreasing contractile function (1, 2). Delivery of adult stem cells wit myogenic potential could overcome both increased muscle fragility by restoring dystrophin expression and decreased regeneration by providing an exogenous source of regenerative potential. While several cell sources have been proposed, all have had significant setbacks owing either to poor engraftment and functional recovery or inability to isolate sufficient numbers of cells for clinical translation. However, adipose-derived stem cells (ASCs) can be derived in large numbers from a relatively expendable tissue and have shown some myogenic potential (26, 29). However, as ASCs are known to be highly adipogenic in-vivo and fatty replacement of muscle tissue is a hallmark of DMD, it is critical to ensure that they remain myogenic upon injection and do not contribute to the formation of intramuscular fat. Mechanical induction of myogenesis in ASCs is more robust than chemical and mechanically-induced myotubes retain their fused state when switched to nonmyogenic conditions. We therefore propose (Aim 1.a) to understand the critical differences between how ASCs and the resident source of intramuscular fat, fibro/adipogenic progenitors (FAPs), sense and respond to mechanical differentiation cues; (Aim 1.b) to exploit those differences to mechanically-condition ASCs away from the FAP phenotype to become and remain myogenic even in the face of adipogenic cues; and (Aim 2) to perform intramuscular injections of ASC-derived myotubes in dystrophic muscle with faty infiltration and ases engraftment, dystrophin expression, and restoration of degenerated muscle function. Successful validation of functional muscle restoration using ASC-derived myotubes and exploration of the mechanisms behind mechano-sensitive fusion will lead to larger animal studies and potential clinical translation. ! PUBLIC HEALTH RELEVANCE: Duchenne Muscular Dystrophy (DMD) is a debilitating disorder that afects 1 in every 3,600 male births and the early onset and severity of DMD significantly impact the quality of life both for patients and families. There is currently no curefor DMD, but cell-based therapies focused on circumventing the progressive muscle wasting at the heart of the disorder hold tremendous promise for improving patient outcomes. We will show that adipose-derived stem cells are a clinically viable cell source for promoting muscle regeneration and improving contractile performance in dystrophic muscle.